Ishibashi Shoko, Love Nick R, Amaya Enrique
The Healing Foundation Centre, The Faculty of Life Sciences, University of Manchester, Manchester, England, UK.
Methods Mol Biol. 2012;917:205-18. doi: 10.1007/978-1-61779-992-1_12.
Here we present a protocol for generating transgenic embryos in Xenopus using I-SceI meganuclease. This method relies on integration of DNA constructs, containing one or two I-SceI meganuclease sites. It is a simpler method than the REMI method of transgenesis, and it is ideally suited for generating transgenic lines in Xenopus laevis and Xenopus tropicalis. In addition to it being simpler than the REMI method, this protocol also results in single copy integration events rather than tandem concatemers. Although the protocol we describe is for X. tropicalis, the method can also be used to generate transgenic lines in X. laevis. We also describe a convenient method for designing and generating complex constructs for transgenesis, named pTransgenesis, based on the Multisite Gateway(®) cloning, which include I-SceI sites and Tol2 elements to facilitate genome integration.
在此，我们展示了一种使用I-SceI巨核酸酶在非洲爪蟾中生成转基因胚胎的方案。该方法依赖于包含一个或两个I-SceI巨核酸酶位点的DNA构建体的整合。它是一种比REMI转基因方法更简单的方法，非常适合在非洲爪蟾和热带爪蟾中生成转基因品系。除了比REMI方法更简单之外，该方案还会导致单拷贝整合事件而非串联多联体。尽管我们描述的方案是针对热带爪蟾的，但该方法也可用于在非洲爪蟾中生成转基因品系。我们还描述了一种基于多位点Gateway(®)克隆设计和生成用于转基因的复杂构建体的便捷方法，称为pTransgenesis，其包括I-SceI位点和Tol2元件以促进基因组整合。
